Abstract: Botulinum Neurotoxins (BoNTs) are a large family of protein toxins and are of great significance due to their extreme potency and the severity of the disease they cause in humans and animals. Botulism is a neuroparalytic disease of long duration, lasting up to several months. Without proper medical care, naturally occurring botulism is lethal in up to 50% of cases, and even with respiratory and other supportive care and antitoxin administration, up to 5 % of patients die. While naturally occurring botulism is rare, BoNTs are classified as a Tier 1 Category A Select Agent due to their threat as potential bioterrorist weapons. Amazingly, BoNTs are also widely used in medicine to treat more than 100 neuromuscular disorders and for aesthetic purposes, which is now an over 2 billion dollar industry and is growing. BoNTs are immunologically divided into 7 serotypes, which are further subdivided into subtypes. Today, 100s of BoNT variants have been identified by sequencing efforts, but only few have been investigated at the protein level. It is remarkable that out of all these BoNT variants only two are currently used in the medical field, and studies examining benefits of using other variants are rare. Our RO1 project `Analysis of BoNT/A subtypes' has determined for the first time that subtypes within the BoNT/A serotype have distinct biologic properties, including cell entry kinetics, duration of action, potency, and immunological variations. This renewal project proposes to extend these data to determine on a molecular and structural level the mechanisms underlying these unique properties. Specifically, this project will investigate the mechanisms underlying the shorter duration of action of BoNT/A3, the faster and more efficient cell entry by BoNT/A2, and the 1,000 fold lower potency of BoNT/A4. Our collaborative efforts are unique in this area, as we are able to combine detailed mechanistic studies on subdomains with cell-based and in vivo studies on the holotoxin level. The Barbieri laboratory will use structural modeling to guide extensive mutagenesis studies on functional domains of BoNTs and investigate mechanisms of receptor binding and cell trafficking using neuronal cell models. The Johnson/Pellett laboratory will utilize these data to create targeted holotoxin constructs in their native hosts and conduct detailed investigations in various cell models, including human cell models. Finally, based on the data from these studies, select holotoxin constructs will be investigated in mice to determine the pathologic and pharmacologic consequences of structural alterations. Using this approach, we are able to investigate a large number of amino acid substitutions and select specific alterations for the more effort- and cost-intensive construction of recombinant holotoxins. By utilizing several cell models before conducting in vivo studies, we are able to reduce the number of required animals and also use human specific models. Finally, the combination of in vitro, cell-based, and in vivo studies will provide novel insight into the mechanisms underlying the observed pathologic and pharmacologic properties of these toxins.